The principal objective of the proposed research is the identification of the mechanistic basis for a progressive neurological disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), which involves intention tremor, gait ataxia, and dementia, and affects at least 1/3 of males over 50 years of age who carry small (premutation, CGGrepeat) expansions of the fragile X mental retardation 1 (FMR1) gene. The neuropathological hallmark of FXTAS is the presence of intranuclear neuronal and astrocytic inclusions, found in the brains of all individuals examined to date who had suffered from the neurodegenerative disorder. The inclusions are ubiquitin-positive, and possess the shape and range of sizes found with the polyglutamine (GAG repeat) disorders. However, there is no known protein abnormality associated with carriers of premutation alleles. The apparent absence of this disorder among males with full mutation alleles (>200 repeats), whose FMR1 gene is generally silenced, coupled with substantially elevated levels of FMR1 mRNA among the premutation carriers, has led to the central hypothesis of this proposal, namely, that the neurological disorder is the consequence of a "toxic" gain-of-function of the FMR1 mRNA. The first 2 aims of this project are to identify the proteins and RNA species that are contained within purified populations of inclusions. These aims will be met through a combination of state-of-the-art mass spectroscopic analysis of protein/peptide identities, and detailed probing of RNA species now known to exist within the inclusions. The third aim of this project will be to determine the potential functional significance of the proteins/RNA species identified under the first 2 aims. Aim 4, a major component of the proposed research, will be to further define the nature of the molecular dysregulation that leads to both inclusion formation and FXTAS, using primary, cultured astrocytes (already established in the laboratory) from both normal individuals and premutation carriers. This neural cell system will be used to define the factors and temporal sequence of events leading to inclusion formation. Several features of the intranuclear inclusions associated with FXTAS are shared with the cytoplasmic inclusions found in Parkinson's disease and the Lewy body dementias, and the glial cytoplasmic inclusions found in multiple system atrophy. Thus, knowledge of the mechanisms leading to the inclusions in FXTAS, as a single-gene disorder, should lead to a broader understanding of the events leading to inclusion formation in other neurodegenerative disorders.